As the ultimate regulator of reproductive function, Gonadotropin-releasing hormone (GnRH) is at the apex of the hypothalamic-pituitary-gonadal axis. It controls puberty, the menstrual cycle, fertility, menopause, and when disregulated, causes infertility. GnRH is secreted in a pulsatile pattern from a population of ~800 neurons, scattered throughout the hypothalamus, to regulate gonadotropins in the pituitary. The overall goal of this renewal application is to elucidate the molecular mechanisms that regulate GnRH in hypothalamic neurons at the levels of gene expression and pulsatile release, both in vitro and in vivo. We will utilize two major model systems: our immortalized GnRH-secreting hypothalamic cells (GT1) and genetically manipu- lated mice. Astonishingly, GT1 cells secrete GnRH in a pulsatile fashion with a 30 min periodicity mirroring mouse GnRH neurons in vivo. We have shown that this rhythm, as well as the estrous cycle, are dependent upon the circadian transcriptional loop. Furthermore, we have identified enhancer regions that target GnRH gene expression exclusively to cultured GT1 cells and to GnRH neurons in vivo. In addition, we have shown that the transcriptional regulators Dlx,Msx, Pbx, Prep, Meis, Oct-1, NF-1, GATA-4, Otx-2, and the Grg co- repressors act to control GnRH gene expression in vivo and in GT1 cells. We propose three aims: Aim 1 will address regulation of the GnRH gene. Utilizing bioinformatics, we have identified additional upstream regula- tory regions and their roles in transcriptional control of the GnRH gene will be determined. Chromatin struc- ture and its role in differential regulation will be examined. Our goals include addressing the hypothesis that MAGE proteins lost in Prader-Willi Syndrome act to relieve Msx repression of the GnRH gene potentially explaining infertility in these patients. Aim 2, will address the roles of Otx-2 and GATA-4 in development using GT1 cells in culture and GnRH-neuron-specific knock-out mice. In Aim 3, we test the hypothesis that the GnRH neuron is a direct target of steroid hormones as well as responsive to afferent input from steroid- sensitive interneurons. We support this aim with preliminary data that GnRH pulses from GT1 cells are regulated by estrogen and progesterone in vitro. Overall, this proposal will address the molecular basis for the developmental regulation of GnRH gene expression and the mechanisms responsible for steroid modulation of GnRH gene expression and pulsatile secretion during the estrous cycle in vivo and in vitro.